Our long term objective is to come to an understanding of the development and organization of the retina, its photoreceptor cells and the neural networks involved in the transduction of visual information. The genome contains most of the instructions that direct the complex control of gene expression, morphogenetic movements of cells and the trafficking of gene products within cells, all of which contribute to development. The aim of the proposed research project is to relate the molecular expression of genes important for eye development to the cell biology of the developing photoreceptor neurons. The photoreceptor cells of the adult compound eye of Drosophila are excellent material for such a study because: 1) they are extremely polarized with a cylindrical soma extending up to 100 mum and axons projecting to precise cell-cell contacts deep in the first and second optic lobes, 2) a host of mutants have been identified which affect these cells, 3) numerous cloned DNAs are available which are expressed in the retina. The molecular and cell biological mechanisms that contribute to the development of the fly's retina may represent general mechanisms of developmental control for other species, including humans. Recently, the use of techniques to localize the expression of transcripts in situ has revealed that certain mRNAs become localized subcellularly. The shuttling of gene products to particular regions of a cell to establish functional polarity has long been investigated at the protein level. The sorting of particular mRNA transcripts may be of equal importance for establishing cell polarity and other cellular functions. To study this aspect of the molecular cell biology of photoreceptor development, higher resolution is required. We propose to merge techniques, already successfully used to localize endogenous mRNAs within tissue sections for light microscopy, with techniques for localizing antigens in ultrastructurally preserved tissue for high resolution electron microscopy. Biotin-labeled DNA probes will be hybridized in situ to ultra-cryo sectioned, aldehyde-fixed tissue. Subsequent to washings,the biotin will be immunohistochemically localized with colloidal gold-conjugated antibodies. The combination of these two approaches will allow us to study the expression and subcellular localization of mRNA transcripts while maintaining ultrastructural resolution. Developing this technique will be extremely useful for further studies investigating the mechanisms for subcellular localization of particular mRNA transcripts. The proposed research will investigate the cellular controls on the expression of the neurodeterminative protein, sevenless, as compared with the photoreceptor specific cell adhesion protein, chaoptic and function receptor protein, opsin DmRh1.